For more than a decade, adenovirus (Ad) serotype 5-based vectors have been used in gene therapy, with limited success. The main disadvantages of Ad5-based vectors appear to be preexisting immunity in the majority of humans, low transduction of important gene therapy target cells (due to low expression of the primary Ad5 receptor, CAR), and innate toxicity induced upon intravascular application (in part due to uptake by cells of the reticuloendothelial system, particularly Kupffer cells). Recent data indicate that vectors containing fibers from Ad group B serotypes 11 and 35 recognize cellular receptors different from CAR and efficiently transduce human cell types that are relatively refractory to Ad5 infection, including malignant tumor cells, bone marrow derived hematopoietic stem cells, mesenchymal cells, and dendritic cells. Furthermore, we found that intravenously injected vectors containing B-group Ad35 or Ad 1 fibers only inefficiently transduce Kupffer cells and therefore elicit significantly reduced innate toxicity as compared to Ad5 vectors. However, little is known about mechanisms and effects of B-group Ad vector infection in vitro and in vivo. Before a clinical application of these new vectors can be considered, this knowledge gap has to be filled. Therefore, the central goal of this proposal is to study the efficiency, specificity, and safety of B-group Ad vector infection on the cellular and organism levels. We will first delineate the cellular receptors involved in B-group Ad infection and construct a set of reporter vectors containing fibers from representative B-group Ad serotypes that differ in the usage of cellular receptors. To study the effects of Ad infection on cell biology (including signaling and changes in gene expression), we will focus on human cell cultures that express B- group Ad receptors and are relevant targets for ex vivo gene therapy. Then, upon in vivo application, we will study the biodistribution of B-group Ad vectors and monitor blood parameters, histopathology, cytokine production/release, and anti-vector immune responses. These studies will be performed in transgenic mice that express B-group Ad receptors. Based on this, we will select representative vectors and parameters and repeat biodistribution and safety studies in baboons.